Two different families of reagents that improve primer specificity when added to a PCR amplification have been described by our laboratory. One family of reagents, described in international patent application WO 2006/044995, is hairpin shaped single stranded oligonucleotides modified at both the 3′ and 5′ ends so that the end of the stem is stabilized relative to a DNA-DNA hybrid, such as by addition of dabcyl moieties, by addition of Black Hole Quencher™ moieties, or by inclusion of 2′ O-methyl nucleotides at the end of the stem. The stem-loop oligonucleotides have loops of 3-22 nucleotides and stems having calculated Tms of 50-85° C. An example of such a reagent is dabcyl-CATTATAATGAAATTATAGTA-dabcyl (SEQ ID No. 1), where the complementary nucleotides of the nine-nucleotide-long stem are underlined. Among the molecules tested for activity reported in WO 2006/044995 were hairpin shaped molecules having fluorescent FAM moieties on both the 3′ and 5′ ends of the stem. These molecules were not active in suppressing mis-priming, even at 1000 nM. It was concluded that “Adding a pair of FAM fluorophores, which are not believed to interact with one another in a stem-stabilizing fashion, was destabilizing, as was adding a single 5′ Dabcyl.” (WO 2006/044995 at page 31, lines 17-19).
The second family of reagents for suppression of mis-priming, described in international patent application WO 2010/105074, is pairs of complementary or partially complementary oligonucleotides that form a hybrid 6-50 nucleotides long, wherein the oligonucleotides are modified on one or both ends by addition of polycyclic moieties, for example, dabcyl moieties, that do not have bulky portions that are non-planar. Bulky non-planar groups, such as fluorescein (FAM) were tested and judged to be not useful as modifying groups in PCR additives. WO 2010/10507 at [0114]).